The Eph subfamily represents the largest known group of receptor-type tyrosine kinases. The extracellular domains of the members of the Eph subfamily contain motifs characteristic of cell adhesion molecules, while the cytoplasmic domains comprise typical tyrosine kinase catalytic domains. The Eph-related kinases are preferentially expressed during embryonic development rather than in adult tissues. A common characteristic of many Eph-related kinases is the predominant expression in the nervous system. Presumably the Eph-related kinases are all derived from the same ancestral gene, and as members of a gene family they share common general characteristics. Different members of the Eph subfamily presumably carry out related, but specialized functions. The widespread expression, distinctive tissue distribution and developmental regulation of the kinases of the Eph subfamily suggest multiple key roles in development. The broad long term objectives of the proposed research are to elucidate the developmental roles of the Eph gene family as a whole and identify the specialized roles of individual Eph-related kinases by using a variety of different molecular approaches. The specific aims of this proposal will focus on Cek9 as the prototype of the Eph subfamily. Cek9 is developmentally regulated, expressed in embryonic neural as well as non- neural tissues, but undetectable in most adult tissues, with the exception of the thymus, retina and brain. Its expression in developing neuronal processes suggests that Cek9 may be important in axon growth or pathfinding. A distinctive characteristic of Cek9 compared to other Eph- related kinases is its ubiquitous phosphorylation on tyrosine in chicken embryonic tissues during specific developmental stages. Since enhanced phosphorylation on tyrosine of Eph-related kinases is indicative of catalytic activation, its in vivo phosphorylation suggests that Cek9 is highly active and thus plays an important role during development. The expression pattern of Cek9, and its alternatively spliced and phosphorylated forms, will be further characterized and compared to that of potential ligand(s) and substrates. The molecular components of the signaling pathways in which Cek9 is implicated will be dissected by searching for the molecules that functionally interact with Cek9. The Cek9 ligand(s) and cytoplasmic targets which are identified will also be examined for their ability to interact with other Eph-related kinases. Finally, the developmental role of Cek9 will be investigated by gene knock out through homologous recombination. These studies will elucidate whether abnormalities in the Eph subfamily of tyrosine kinase genes are likely to be the cause of any human congenital disease or developmental pathology. The identification of the cellular processes that are regulated by the Eph-related kinases, which may include cell fate determination, cell proliferation and differentiation, nerve cell survival and regeneration, and axon growth and pathfinding, will allow the manipulation of such processes with possible therapeutic outcomes.